The invention had the object of finding novel compounds having valuable properties, in particular those which can be used for the preparation of medicaments.
One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are highly regulated and often overlap, as is evident from the existence of many protein kinases as well as phosphatases. Phosphorylation of proteins occurs predominantly at serine, threonine or tyrosine residues, and protein kinases have therefore been classified by their specificity of phosphorylation site, i.e. serine/threonine kinases and tyrosine kinases. Since phosphorylation is such a ubiquitous process within cells and since cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or diseases are attributable to either aberrant activation or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterisation of these proteins and compounds that are able to modulate their activity (for a review see: Weinstein-Oppenheimer et al. Pharma. &. Therap., 2000, 88, 229-279).
The present invention relates to compounds and to the use of compounds in which the inhibition, regulation and/or modulation of signal transduction by protein kinases, in particular tyrosine kinases and/or serine/threonine kinases, plays a role, furthermore to pharmaceutical compositions which comprise these compounds, and to the use of the compounds for the treatment of kinase-induced diseases.
In particular, the present invention relates to compounds and to the use of compounds in which the inhibition, regulation and/or modulation of signal transduction by kinases plays a role, in particular the protein kinases NUAK1, also known as ARKS, MAPK (MAP2K1, MAP4K2, MAP3K11), MARK3, ROCK2, also known as Rho kinase 2, CHEK1, also known as CHK1, CDK2, PKN2, KDR, also known as VEGFR, HIPK1 and AURORA.
MAPxKy (Mitogen-activated Protein (MAP) Kinase
The protein encoded by this gene is a member of the dual-specific protein kinase family which functions as a mitogen-activated protein (MAP) kinase. MAP kinases, also described as extracellular signal-regulated kinases (ERKs), function as integration point for various biochemical signals. MAP2K1 is located in front of other MAP kinases in the signal cascade and stimulates the enzymatic activity thereof as a function of many extra- and intracellular signals. As an essential component of the MAP kinase signal transduction pathway, MAP2K1 is involved in many cellular processes, such as proliferation, differentiation, transcription regulation and cell development.
Similar Considerations Apply to Other MAPKs.
The compound RO-4927350, a thiazole derivative, has been described by K. Kolinsky et al. as MEK inhibitor for the specific inhibition of the MAPK signal transduction cascade in Cancer Res. 2009; 69: 1924 ff., where the compound has an antitumour activity in vivo.
T. Kato et al. in Neoplasia 2001; 3 (1): 4-9, describe MARK3 (homologous to MARKL1) as potential target for the treatment of hepatocellular carcinogenesis.
CHEK1
Studies confirm that CHEK1 inhibition increases the cytotoxicity of DNA-damaging agents (Xiao Z, Chen Z, Gunasekera A H, et al. Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents, J. Biol. Chem. 2003; 278: 21767-73;
Xiao D, Herman-Antosiewicz A, Antosiewicz J, et al. Diallyl trisulfide-induced G(2)-M phase cell cycle arrest in human prostate cancer cells is caused by reactive oxygen species-dependent destruction and hyperphosphorylation of Cdc 25 C, Oncogene 2005; 24: 6256-68;
Zhao B, Bower M J, McDevitt P J, et al. Structural basis for Chk1 inhibition by UCN-01, J. Biol. Chem. 2002; 277: 46609-15;
Maude S L, Enders G H. Cdk inhibition in human cells compromises chk1 function and activates a DNA damage response. Cancer Res. 2005; 65: 780-6.)
and the expression of CHEK1 is part of the defense mechanism of the cell for avoiding the toxicity of DNA damage.
ROCK2
ROCK2 is a serine/threonine kinase which is activated by association with RhoGTP, which results in phosphorylation of a multiplicity of substrates and ultimately results in stabilisation of filamentous actin and an increase in the activity of myosine ATPase. This in turn causes the formation of contractile actin-myosine units (stress fibres) and integrin-containing focal adhesions. Through the modulation of actin-myosine contractility, ROCK2 has a significant influence on the regulation of cell morphology, cell mobility and cell adhesion. C. Chak-Lui Wong et al. in Hepatology 2009; 49 (5): 1583-94, describe that the inhibition of Rho kinases 1 and 2 (ROCK1 and ROCK2) can be utilised for the treatment of cancer diseases. Thus, ROCK2 plays a significant role in the growth of hepatocellular carcinomas. X. Q. Wang et al. in Radiation Res. 2007; 168 (6): 706-15, describe the checkpoint kinase CHK1 as potential target for the treatment of cancer.
NUAK1
The Nuak1 gene encodes for the “NUAK family SNF1-like kinase 1”. NUAK1 interacts with USPX9 and ubiquitin C.
The role of NUAK1 (ARK5) as growth or nutrition factor of tumour cells has been described by A. Suzuki et al. in J. Biol. Chem. 2003; 278 (1): 48-53, and in Oncogene 2003; 22: 6177-82.
The inhibition of NUAK1 (ARK5) thus represents a potential possibility for combating cancer
CDK2
The deactivation of CDK2 prevents the phosphorylation of the protein RB1.
The cell is thus unable to leave the G1 phase of the cell cycle, which results in stopping of all cell division.
J. K. Buolamwini in Current Pharmaceutical Design 2009; 6, (4): 379-92, describes the therapeutic potential of CDK (cyclin dependent kinase) inhibitors in the combating of cancer.
A. Schmidt et al. in EMBO J. 2007; 26: 1624-36, describe PRK2/PKN2 as potential target for the treatment of tumour diseases.
N. Ferrara in Endocrine Rev. 2004; 25 (4): 581-611, describes the use VEGF inhibitors for combating cancer.
VEGF and KDR represent a ligand-receptor pair which plays an essential role in the proliferation of vascular endothelial cells and in the formation and sprouting of blood vessels, which are known as vasculogenesis or angiogenesis.
Angiogenesis is characterised by above-average activity of vascular endothelial growth factor (VEGF). VEGF actually consists of a family of ligands (Klagsburn and D'Amore, Cytokine & Growth Factor Rev. 1996; 7: 259-70,). VEGF binds the high-affinity membrane-spanning tyrosine kinase receptor KDR and the related fms tyrosine kinase-1, also known as Flt-1 or vascular endothelial cell growth factor receptor 1 (VEGFR-1). Cell culture and gene knockout experiments indicate that each receptor contributes to different aspects of angiogenesis. KDR mediates the mitogenic function of VEGF, whereas Flt-1 appears to modulate non-mitogenic functions, such as those associated with cellular adhesion. Inhibition of KDR thus modulates the level of mitogenic VEGF activity. In fact, it has been shown that tumour growth is influenced by the antiangiogenic action of VEGF receptor antagonists (Kim et al., Nature 1993; 362: 841-4).
Three PTK (protein tyrosine kinase) receptors for VEGFR have been identified: VEGFR-1 (Flt-1); VEGRF-2 (Flk-1 or KDR) and VEGFR-3 (Flt-4).
The fact that the inhibition of VEGFR (vascular endothelial growth factor receptor 2—KDR) is of importance for tumour therapy has been shown by means of the introduced medicaments sunitinib, sorafenib and vatalanib, which also inhibit VEGFR.
HIPK1
HIPK1, a nuclear protein kinase which is present in increased concentrations in breast cancer cells, serves to phosphorylate various transcription factors, including p53.
It can be assumed that HIPK1 plays a role in cancer and tumorigenesis by regulating p53 and/or the Mdm2 function.
Y. Aikawa et al. in EMBO J. 2006; 25: 3955-65, describe the homeodomain-interacting protein kinases HIPK1, HIPK2 and HIPK3 as potential targets for the treatment of tumour diseases.
R. Copeland et al. in FASEB J. 2008; 22: 1050 ff., describe HIPK1 as target for the treatment of cancer diseases.
HIPK2 (homeodomain interacting protein kinase)
The significantly increased expression of HIPK2 in cervical cancer seems to correlate with the progression of the disease (Eva Krieghoff-Henning javascript:popRef(‘a1’) & Thomas G Hofmann Future Oncology 2008; 4 (6): 751-54).
The role of aurora kinase inhibitors in the treatment of tumours are described by a number of authors:
D. S. Boss et al., The Oncologist 2009; 14: 780-93;
S. Lapenna et al., Nature Rev. Drug Discov. 2009; 8: 547-66; 35
L. Garuti et al., Cur. Med. Chem. 2009; 16, 1949-63;
J. R. Pollard et al., J. Med. Chem. 2009; 52 (9): 2629-51;
C. H. A. Cheung et al., Expert Opin. Investig. Drugs 2009; 18 (4): 379-98.
The present invention therefore relates to the use of the compounds of the formula I for the treatment of diseases or conditions in which inhibition of the activity of protein kinases, in particular the protein kinases NUAK1, also known as ARK5, MAPK (MAP2K1, MAP4K2, MAP3K11), MARK3, ROCK2, also known as Rho kinase 2, CHEK1, also known as CHK1, CDK2, PKN2, KDR, also known as VEGFR, HIPK1 and AURORA, is advantageous.
The synthesis of small compounds which specifically inhibit, regulate and/or modulate signal transduction by tyrosine kinases and/or serine/threonine kinases, in particular the above-mentioned protein kinases, is therefore desirable and an aim of the present invention.
It has been found that the compounds according to the invention and salts thereof have very valuable pharmacological properties while being well tolerated.
The present invention specifically relates to compounds of the formula I which inhibit, regulate and/or modulate signal transduction by protein kinases, to compositions which comprise these compounds, and to processes for the use thereof for the treatment of kinase-induced diseases and complaints, such as angiogenesis, cancer, tumour formation, growth and propagation, arteriosclerosis, ocular diseases, such as age-induced macular degeneration, choroidal neovascularisation and diabetic retinopathy, inflammatory diseases, arthritis, thrombosis, fibrosis, glomerulonephritis, neurodegeneration, psoriasis, restenosis, wound healing, trans-plant rejection, metabolic diseases and diseases of the immune system, also autoimmune diseases, cirrhosis, diabetes and diseases of the blood vessels, also instability and permeability and the like in mammals.
Solid tumours, in particular fast-growing tumours, can be treated with kinase inhibitors. These solid tumours include monocytic leukaemia, brain, urogenital, lymphatic system, stomach, laryngeal and lung carcinoma, including lung adenocarcinoma and small-cell lung carcinoma.
The compounds of the formula Ia and Ib can furthermore be used to provide additive or synergistic effects in certain existing cancer chemotherapies, and/or can be used to restore the efficacy of certain existing cancer chemotherapies and radiotherapies.
It can be shown that the compounds according to the invention have an antiproliferative action in vivo in a xenotransplant tumour model. The compounds according to the invention are administered to a patient having a hyperproliferative disease, for example to inhibit tumour growth, to reduce inflammation associated with a lymphoproliferative disease, to inhibit trans-plant rejection or neurological damage due to tissue repair, etc. The present compounds are suitable for prophylactic or therapeutic purposes. As used herein, the term “treatment” is used to refer to both prevention of diseases and treatment of pre-existing conditions. The prevention of proliferation is achieved by administration of the compounds according to the invention prior to the development of overt disease, for example to prevent the growth of tumours, prevent metastatic growth, diminish restenosis associated with cardiovascular surgery, etc. Alternatively, the compounds are used for the treatment of ongoing diseases by stabilising or improving the clinical symptoms of the patient.
The host or patient can belong to any mammalian species, for example a primate species, particularly humans; rodents, including mice, rats and hamsters; rabbits; horses, cows, dogs, cats, etc. Animal models are of interest for experimental investigations, providing a model for treatment of human disease.
The susceptibility of a particular cell to treatment with the compounds according to the invention can be determined by in vitro tests. Typically, a culture of the cell is combined with a compound according to the invention at various concentrations for a period of time which is sufficient to allow the active agents to induce cell death or to inhibit migration, usually between about one hour and one week. In vitro testing can be carried out using cultivated cells from a biopsy sample. The viable cells remaining after the treatment are then counted.
The dose varies depending on the specific compound used, the specific disease, the patient status, etc. A therapeutic dose is typically sufficient considerably to reduce the undesired cell population in the target tissue while the viability of the patient is maintained. The treatment is generally continued until a considerable reduction has occurred, for example an at least about 50% reduction in the cell burden, and may be continued until essentially no more undesired cells are detected in the body.
For identification of a signal transduction pathway and for detection of interactions between various signal transduction pathways, various scientists have developed suitable models or model systems, for example cell culture models (for example Khwaja et al., EMBO J. 1997; 16: 2783-93) and models of transgenic animals (for example White et al., Oncogene 2001; 20: 7064-72). For the determination of certain stages in the signal transduction cascade, interacting compounds can be utilised in order to modulate the signal (for example Stephens et al., Biochemical J. 2000; 351: 95-105). The compounds according to the invention can also be used as reagents for testing kinase-dependent signal transduction pathways in animals and/or cell culture models or in the clinical diseases mentioned in this application.
Measurement of the kinase activity is a technique which is well known to the person skilled in the art. Generic test systems for the determination of the kinase activity using substrates, for example histone (for example Alessi et al., FEBS Lett. 1996; 399 (3): 333-8) or the basic myelin protein, are described in the literature (for example Campos-González, R. and Glenney, Jr., J. R. J. Biol. Chem. 1992; 267: 14535).
For the identification of kinase inhibitors, various assay systems are available. In scintillation proximity assay (Sorg et al., J. Biomol. Screen. 2002; 7: 11-19) and flashplate assay, the radioactive phosphorylation of a protein or peptide as substrate with γATP is measured. In the presence of an inhibitory compound, a decreased radioactive signal, or none at all, is detectable. Furthermore, homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET) and fluorescence polarisation (FP) technologies are suitable as assay methods (Sills et al., J. Biomol. Screen. 2002; 7 (3): 191-214).
Other non-radioactive ELISA assay methods use specific phospho-anti-bodies (phospho-ABs). The phospho-AB binds only the phosphorylated substrate. This binding can be detected by chemiluminescence using a second peroxidase-conjugated anti-sheep antibody (Ross et al. Biochem. J. 2002, 366: 977-981).
There are many diseases associated with deregulation of cellular proliferation and cell death (apoptosis). The conditions of interest include, but are not limited to, the following. The compounds according to the invention are suitable for the treatment of various conditions where there is proliferation and/or migration of smooth muscle cells and/or inflammatory cells into the intimal layer of a vessel, resulting in restricted blood flow through that vessel, for example in the case of neointimal occlusive lesions. Occlusive graft vascular diseases of interest include atherosclerosis, coronary vascular disease after grafting, vein graft stenosis, peri-anastomatic prosthetic restenosis, restenosis after angioplasty or stent placement, and the like.